Enzymes containing mononuclear non-heme iron sites catalyze a diverse array of reactions that are significant to medicine and to the environment. The studies described in this proposal focus on the largest group of non-heme iron enzymes, the Fe(ll)- and alpha-ketoglutarate (aKG)-dependent hydroxylases. We seek to better define the hydroxylase reaction intermediates, enhance our understanding of the protein features involved in substrate recognition, and expand our knowledge of the functional roles of related family members. The specific aims include: (1) Use variants of the best-studied representative of this enzyme family, the sulfonate-metabolizing enzyme TauD, to spectroscopically analyze the Fe(IV)-oxo intermediate, examine other catalytic species, and to determine mutant protein structures. Also, investigate TauD interactions with inhibitors and study quantitatively the self-hydroxylation chemistry of this enzyme to test hypotheses regarding the role of the enzyme side chain modification reactions. (2) Elucidate the interactions of the E. coli DMA-repair enzyme AlkB with its substrate, methylated DNA, and investigate the roles of several human homologues. (3) Structurally and spectroscopically characterize the herbicide-degrading enzyme TfdA, and determine the basis for the opposite enahtiospecificities of two related enzymes, RdpA and SdpA, by using structural and mutagenesis approaches. (4) Define the structure, biochemical properties, and spectroscopically accessible catalytic intermediates of a newly identified Fe(ll)/aKG-dependent hydroxylase that oxidizes xanthine. (5) Identify the function of the E. coli Gab protein and spectroscopically examine its catalytic intermediates. (6) Explore the potential for carefully selected Fe(ll)/aKG hydroxylase family members to function in criromatin demethylation. The first three aims continue ongoing investigations in the laboratory, while the latter three aims present new research directions. Insights gained from these studies will be useful in understanding the substrate recognition features and chemical mechanisms of a large number of enzymes, including many less tractable examples that have direct medical relevance.